1. Field of the Invention
The present invention relates generally to the fields of bacteriology and mycology. More particularly, the present invention provides methods and compositions for increasing the effectiveness of existing antibiotics and antifungal agents and methods of overcoming bacterial and fungal resistance.
2. Description of Related Art
Gram positive organisms, particularly Staphylococci, Streptococci, and Enterococci, are increasingly seen as the major aetiological agents in infectious diseases. In the hospital setting, Staphylococcus aureus and Enterococcus faecalis account for more than 50% of isolates from blood stream infections (Cormican and Jones, 1996). In community-acquired infections Streptococcus pneumoniae remains a leading cause of illness and death (Centers for Disease Control, 199). The ongoing and rapid emergence and spread of antibiotic resistance in these organism is thus a problem of crisis proportions.
One of the major impediments in treating Gram-positive infections is their limited susceptibility to fluoroquinolones, the latest addition to the arsenal of antibiotics. Since their introduction in the mid-1980s, fluoroquinolone antibiotics, have become the most used class of antibiotics in the world (Acar and Goldstein, 1997). One such antibiotic., ciprofloxacin (Davis et al., 1996), accounts for 90% of all quinolones used in medicine, Because of its spectrum of activity, oral availability, and relatively low cost. ciprofloxacin has been used for treating a wide range of infections, including those of unknown etiology. In 1996, three new indications for the use of ciprofloxacin were approved suggesting that the use of this antibiotic will continue for many years to come.
Although being highly active against most Gram negative microorganisms (MIC.sub.90 in the range of 0.1 .mu.g/ml), ciprofloxacin is less effective against Gram positive infections, particularly aerobic Gram positive cocci. The MIC.sub.90 values for S. aureus, E. faecalis and S. pneumoniae are in the range of 1-5 .mu.g/ml, whereas the achievable tissue concentration of ciprofloxacin is only 4 .mu.g/ml (Davis et al., 1996). The high intrinsic resistance to ciprofloxacin, and the extensive use of quinolones both in human and veterinary medicine, has led to the emergence and dissemination of ciprofloxacin-resistant Gram-positive strains. This limitation has led to the quest for new, more effective fluoroquinolones.
Antibiotic resistance is mediated, at least in part, by the efflux of drugs from target cells by multidruy transporters (MDTs). These transporters promote the active efflux of a wide variety of drugs, including fluoroquinolone antibiotics, from the bacterial cells that are responsible for the particular infection. In 1991, Neyfakh et al. published the first description of a chromosomally-encoded bacterial multidrug transporter, Bmr, of the Gram positive bacteria Bacillus subtilis. Since then, practically every bacterial species analyzed, including pathogenic species such as Escherichia, Pseudomonas, Mycobacteria, etc. (Lomovskaya and Lewis, 1992; Poole et al., 1993; Takiff et al., 1996, reviewed in Nikaido, 1994; Lewis, 1994), has been shown to express one, or even several multidrug transporters. For example, B subtilis expresses at least three multidrug transporters, homologous Bmr and Blt (Ahmed et al., 1995) and an evolutionarily more distant Bmr3 (Ohki and Murata, 1997). Bmr and its close homolog in Staphylococcus aureus, NorA, promote the efflux of a variety of bacteriotoxic compounds, including ethidium bromide, rhodamine, acridines, tetraphenylphosphonium and puromycin, with fluoroquinolone antibiotics being one of the best transporter substrates (Yoshida et al., 1990; Neyfakh, 1992; Neyfakh et al., 1993). Importantly, drug efflux mediated by the Bmr and NorA transporters can be completely inhibited by the plant alkaloid reserpine, which by itself is not toxic to bacteria (Neyfakh et al.; Neyfakh, 1993).
Multidrug transporters also play an important role in both the intrinsic and acquired resistance of important fungal pathogens to antifuingal agents. Particularly, multidrug transporters contribute to the resistance of Candida albicans, the fourth leading cause of all hospital-acquired infections, to azole antifungal agents.
There is little knowledge regarding the physiological role of multidrug transporters or the mechanism of their action; nevertheless these transporters appear to play an important role in the intrinsic resistance of bacterial cells to toxins and antibiotics. Inactivation of the chromosomal transporter genes usually leads to a dramatic increase in the sensitivity of bacteria to the transporter substrates (Poole et al., 1993; Ahmed et al., 1994; Okusu et al., 1996; Yamada et al., 1997). Disruption of the Bmr gene in B. subtilis, or the inhibition of the Bmr transporter with reserpine, reduces the minimal inhibitory concentration (MIC) of norfloxacin, a typical fluoroquinolone antibiotic, by a factor of five (Neyfakh, 1992). Similarly, multidrug transporters contribute significantly to the intrinsic fluoroquinolone resistance of Gram positive pathogenic cocci. Yamada et al. (1997) have recently shown that genetic disruption of the NorA gene increases the susceptibility of S. aureus to norfloxacin and ciprofloxacin by eight and four fold, respectively. Reserpine, which inhibits NorA-mediated drug efflux, reduces the MIC of norfloxacin for wild-type S. aureus by at least four-fold (Markham and Neyfakh, 1996; Kaatz and Seo, 1995). Although the multidrug transporter of S. pneumoniae has not yet been identified, its existence is strongly supported by physiological data (Baranova and Neyfakh, 1997; Zeller et al., 1997; Brenwald et al.; 1997). Furthermore, reserpine has been shown to reduce the MIC of norfloxacin and ciprofloxacin for wild-type S. pneumonae by the factor of 2-3 (Baranova and Neyfakh, 1997). In E. faecalis the active efflux of fluoroquinolones has been demonstrated biochemically (Lynch et al., 1997) and, again, reserpine provides a two-fold increase in their susceptibility to fluoroquinolones.
In addition to being involved in the intrinsic resistance of Gram-positive cocci to fluoroquinolones, multidrug transporters contribute to the acquired resistance, which is selected upon exposure to these antibiotics. In S. aureus and S. pneumoniae, the acquired resistance has so far been attributed mainly to the sequential acquisition of mutations in the targets of fluoroquinolone action, topoisomerase IV and DNA gyrase (Cambau and Gutman, 1993; Ferrero et al., 1994; Munoz and De La Campa, 1996; Tankovi, 1996). From the limited studies of fluoroquinolone resistance mechanisms in E. faecalis, it appears that mutations of gyrase are present in at least some high levet resistant isolates Korten et al., 1994). However, it has become apparent in recent years that these mechanisms of acquired resistance are complemented by over-cxpression of multidrug transporters. Such overexpression can result from either amplification of the transporter gene (Neyfakh, 1991); or mutations in the regulatory regions of these genes or regulatory proteins controlling their transcription (Ahmed et al., 1995; Kaatz and Seo, 1995).
Overexpression of the NorA multidrug transporter has been reported for strains of S. aureus selected for fluoroquinolone resistance both in vitro (Yoshida et al., 1990; Kaatz et al., 1990) and in vivo (Trucksis et al., 1991). From the discussion above it is clear that multidrug transporters present a major impediment to the treatment of Gram positive pathogenic insult. There exists a need for drug(s) that may circumvent these transporters to be useful in treatment regimens.